Hindawi Publishing Corporation Scienti�ca Volume 2012, Article ID 152365, 15 pages http://dx.doi.org/10.6064/2012/152365

Review Article Genetic Aspects of Congenital and Idiopathic Scoliosis

Philip F. Giampietro

Waisman Center, University of Wisconsin-Madison, 1500 Highland Avenue, Madison, WI 53705, USA

Correspondence should be addressed to Philip F. Giampietro; [email protected]

Received 10 October 2012; Accepted 11 November 2012

Academic Editors: F. Acosta, T. M. George, and S. Rasmussen

Copyright © 2012 Philip F. Giampietro. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Congenital and idiopathic scoliosis represent disabling conditions of the spine. While congenital scoliosis (CS) is caused by morphogenic abnormalities in vertebral development, the cause(s) for idiopathic scoliosis is (are) likely to be varied, representing alterations in skeletal growth, neuromuscular imbalances, disturbances involving communication between the brain and spine, and others. Both conditions are characterized by phenotypic and genetic heterogeneities, which contribute to the difficulties in understanding their genetic basis that investigators face. Despite the differences between these two conditions there is observational and experimental evidence supporting common genetic mechanisms. is paper focuses on the clinical features of both CS and IS and highlights genetic and environmental factors which contribute to their occurrence. It is anticipated that emerging genetic technologies and improvements in phenotypic strati�cation of both conditions will facilitate improved understanding of the genetic basis for these conditions and enable targeted prevention and treatment strategies.

1. Introduction that 11.1 of 1st degree relatives are affected, compared to 2.4 of 2nd degree, and 1.1 of 3rd degree relatives [1]. Advances in developmental biology have enabled improve- By age 16, 0.6 of affected people will have required active ments in the understanding of spine development and have % provided contributions that enhance our understanding of treatment% with a full-time thoracolumbar-sacral% orthosis genetic and environmental factors that are associated with (TLSO) or surgical% correction with instrumentation [2, 3]. congenital and idiopathic scoliosis. is paper will focus Older IS subclassi�cation is based on the age of presentation on salient features of both forms of scoliosis and highlight categorized as: (1) infantile (birth to age 3 years), (2) juvenile research focusing on genetic and environmental mechanisms (age 3 to 11 years), and (3) adolescent (11 years and older). for their occurrence. ese subclassi�cations are sometimes useful clinically, but have no established genetic basis. Age-speci�c genetic markers have not been identi�ed for IS, and the current �. �e�nitions and �pidemiolog� of Scoliosis concept of scoliosis is that the disorder develops continu- Idiopathic scoliosis (IS) is de�ned by the Scoliosis Research ously between the juvenile years and adolescence. Hence, in Society (http://www.srs.org/) as a lateral curvature of the this paper, the term IS is used in most situations without spine of 10 or greater for which no cause can be determined. attempt to distinguish juvenile and adolescent subtypes. e incidence of IS for treatable curves de�ned as 25 or greater ere is evidence∘ that genetic and environmental factors are likely to play a role in the occurrence of both as described is greater in females than in males with a ratio∘ of 2 : 1, herin, although the mechanism responsible for this is uncer- respectively. Gender differences may underlie scoliotic curve tain at the present time. is rotational deformity is measured progression. in the forward bending position by an inclinometer, and the Congenital scoliosis (CS) is a form of spinal curvature scoliometer as an angle of trunk rotation (ATR). which is due to the presence of an underlying congenital e incidence of idiopathic scoliosis (IS) in the general vertebral malformation (CVM). population ranges from 2 to 3 , varying with the de�nition e estimated frequency of CVM in the general pop- of the magnitude of the curve. Population studies indicate ulation is in the range of 0.13–0.5/1,000 [4]. Vertebral % % 2 Scienti�ca malformations most commonly include hemivertebrae (half FGF, and Wnt signaling gradients, posteriorly regressing of a vertebrae), additional vertebrae, vertebral bar (an abnor- as the embryo elongates along the anterior-posterior axis mality of vertebral separation during development), butter�y, [15, 18, 19] Figure 3. e exposure of cells in the posterior and wedge-shaped vertebrae illustrated in Figures 1 and 2. presomitic mesoderm to high levels of FGF and Wnt activity Vertebral malformations may represent an isolated �nding, enables the maintenance of an undifferentiated state [20, occur in association with other renal, cardiac, or spinal 21]. Below the determination front, cells are capable of cord malformations, or occur as part of an underlying responding to the segmentation clock through the activation syndrome or chromosomal abnormality. Autopsy of fetuses of boundary speci�c genes Mesp2 and Riply [21–23]. Wnt3a with anencephaly and myelomeningocele demonstrates the provides a crucial function in both the clock and wavefront presence of cervical and thoracic CVM, suggesting a related portions associated with somitogenesis and through Msgn1 etiology for both neural tube defects and CVM [5]. plays a major role in the segmentation clock through regu- Frequently encountered syndromes associated with CVM lation of Notch and Wnt signaling pathways [24]. As a result include the following: of active Wnt signaling, active Wnt signaling, Msgn1 and Wnt targets are expressed. A phase lag allows for Msgn1 to activate (i) Alagille syndrome (peripheral pulmonic stenosis, Notch related genes. RA plays an important role in the cholestasis, facial dysmorphism); preservation of spine symmetry through its buffering action (ii) �archo-Levin syndrome (short trunk dwar�sm, multi- of the Le-Right pathway which creates asymmetry through ple vertebral and rib defects with posterior rib fusion); theactionofNodal[25]. Since the majority of patients with (iii) Klippel-Feil syndrome (short neck, low posterior IS exhibit a spinal curve to the right, an underlying defect in hairline, and fusion of cervical vertebrae), hemifacial le-right asymmetry has been hypothesized [26]. microsomia (associated with craniofacial anomalies including microtia); (iv) Goldenhar syndrome (hemifacial microsomia and 4. Teratogens Associated with CVM epibulbar dermoids); and Various maternal exposures during pregnancy including (v) VACTERL syndrome (vertebral malformations, anal alcohol use [27], anticonvulsant medications such as val- atresia, cardiac malformations, tracheo-esophageal proic acid [28–30], hyperthermia [31], maternal insulin- �stula, renal, and radial anomalies, and limb defects). dependent diabetes mellitus, and gestational diabetes [32– 34] have been observed to be associated with the occurrence 3. Vertebral Development and Genes Involved of CVM in animal models and humans. Single nucleotide polymorphisms in glucose metabolizing genes including Vertebral bodies are derived from somites through a recur- GLUT1, HK1, and LEP are postulated to be related to the rent process of budding off from the presomitic mesoderm occurrence of malformations observed in diabetic embry- mediated by cyclical expression of FGF, Wnt, and Notch opathy. e occurrence of reactive oxygen species (ROS) has signaling pathway genes [8]. A “clock and wavefront” model been proposed as a mechanism for altered somitogenesis for somitogenesis was originally proposed by Cooke and in diabetic embryopathy [35]. Mutations in the planar cell Zeeman in 1976 [9]. In this model the “clock” represents an polarity gene, CELSR1 (Caherin, EGF Lag Seven Pass G-Type oscillator which connects presomitic mesodermal cells, and Receptor 1–3), have been identi�ed in patients with either the “wave” represents a region of “rapid cellular change” in neural tube defects or caudal agenesis [36]. Mutations in which transition to somite development occurs, presumably planar cell polarity genes are associated with a shortened mediated by some type of gradient. body axis, widened neural plate, and neural tube defects [37]. A similar mechanism of oscillation amongst members of CVM have been observed in laboratory animals exposed to I the Hes/Her/Hairy family of basic helix-loop-helix (bHLH) (Kr)-blockers (class III anti-arrhythmic agent), zinc de�cient transcriptional repressors has been reported in mice, chicken, diet, the organophosphate pesticide chlopyrifos fumonisins and zebra�sh, providing evidence for conservation of the (environmental toxins produced Fusarium moniliforme (F. oscillator in vertebrates [10–13]. A molecular oscillator regu- verticilliodes), F. proliferatum,andotherFusarium species of lates the Notch, Fgf, and Wnt signaling pathways in which the molds), during pregnancy [38–40]. Notch and Fgf genes oscillate in opposite phase to the Wnt Fish with vertebral deformities and abnormal mechanical genes [14]. Wnt3a signaling mediated by -catenin which vertebral properties were produced following exposure of controls the oscillatory signaling in the Notch pathway [15]. juvenile fourhorn sculpin, Myoxocephalus quadricornis L. Following periodic activation of Notch 1, Notch𝛽𝛽 intracellular to tetrachloro-1, 2-benzoquinone, a component in bleached domain (NICD), the cleaved form of the Notch 1 receptor, kra mill effluents [41]. Exposure to carbon monoxide[42] translocates to the nucleus. NICD activates transcription and boric acid are associated with alterations in HOX- of multiple target genes including Hairy/Hes/Her genes, mediated gene expression [43]. Retinoic acid, a vitamin A Lunatic fringe (Lfng), and Notch-related ankyrn repeat pro- analogue, has been observed to cause homeotic transforma- tein (Nrap) [14, 16, 17]. tions in mice and axial skeletal truncation in the Dominant A stripe of expression of genes occurs in response to the hemimelia (dh) mouse, suggesting a possible relationship periodic clock signal at a region referred to as the determina- between retinoic acid signaling and the dh gene [44]. tion front, which is de�ned by opposing retinoic acid (RA), Increased axial skeletal defects and apoptosis were associated Scienti�ca 3

Congenital scoliosis Defects of formation Hemivertebra Wedge vertebra

Unilateral Unilateral complete failure partial failure of formation of formation

Fully segmented Semi-segmented Incarcerated Nonsegmented Defects of segmentation Unilateral unsegmented bar Unilateral bar and hemivertebra Block vertebra

Unilateral failure of Bilateral segmentation failure of segmentation

F 1: Diagram of spine illustrating defects of formation (wedge and hemivertebrae) and segmentation (vertebral bar and block vertebrae). Reprinted with permission from McMaster [6].

120 mm (191.86) and CVM. Similar to other birth defects, CVM oen repre- 40665:3 sent sporadic occurrences making epigenetic factors another 0 D plausible mechanism for investigation. 7.6 T-spine head first Helical mode Hyperthermia has been associated with CVM devel- opment. Heat shock proteins are recruited when there is exposure to nonteratogenic doses of heat ( 2 C) which provide protection for proteins against subsequent∘ damage by teratogenic doses of heat ( 2 C). Heat shock< proteins attach to uncovered active sites, thus∘ preventing their binding with other functionally impaired> aggregate proteins [51]. Hyperthermia results in inhibition of the cell cycle and F 2: 3D reconstruction illustrating congenital scoliosis. Le T4 induces apoptosis. Although the exact mechanism responsi- hemivertebrae displayed. Courtesy of Dr. Kenneth Noonan. ble for altered somitogenesis associated with heat is uncer- tain, Notch/Delta signaling pathway proteins may undergo alteration(s) and result in abnormal vertebral patterning. Presently there are no reported studies which describe the relative contribution of maternal exposures to CVM with inhibition of nitric oxide (NO) production or the development. In a series of 206, 244 live births, still births, addition of NO to developing chick embryos [45]. Low birth and elective terminations, a total of 5 cases of isolated weight, decreases in successive births, and behavioral de�cits hemivertebrae, and 22 cases of hemivertebrae with other replicated by carbon monoxide alone in animal models have birth defects were identi�ed [52]. e most common mater- been reported to occur in conjunction with cigarette smoking nal exposure associated with CVM was maternal diabetes (5 during pregnancy [46, 47]. Cigarette smoke generation of cases) followed by twinning (2 cases). ROS resulting in somite anoxic damage could potentially contribute to the development of CVM. e occurrence of CS in monozygotic twins [48] is consistent with an observed increased risk for congenital  $MBTTJêDBUJPO PG $7. malformations in both monozygotic and dizygotic twins [49]. Congenital malformations and syndromes including Prader- A classi�cation scheme for CVM which is simple and uni�ed Willi, Angelman, and Beckwith-Wiedemann syndromes have is essential for clinicians and researchers to describe individ- been linked to assisted reproductive technology (ART) [50]. ual and collective CVM from both a phenotypic and genetic Methyl donor content of the growth media has been sug- etiologic vantage point. A number of classi�cation schemes gested as a possible mechanism of CVM occurrence in for CVM have been proposed which have individually ART-assisted pregnancies, and nutritional factors have been focused upon different components associated with CVM, implicated for their occurrence in non-ART pregnancies, an occurrence including a developmental basis for CVM, suggesting a possible relationship between epigenetic factors [53, 54] syndromic diagnosis of CVM (i.e. spondylocostal 4 Scienti�ca

Proposed ICVAS classification scheme for congenital vertebral malformations ICVAS classification (clinical)

VSD

Multiple (MVSD) Single (SVSD)

Defined e.g., Alagille, VATER, etc.

Undefined Generalized (SVSD-U)

Defined MYSD-SCD or STD

Spondylocostal dysostosis

Spondylothoracic dysostosis

Undefined (MVSD-GU)

Regional State whether cervical, dorsal, lumbar or sacral

Defined Undefined Alagille, VATER, VACTERL, (MVSD-RU) Goldenhar, CHARGE, etc

U: Undefined R: Regional G: Generalized

F 3: Algorithm for International Consortium for Vertebral Anomalies and Scoliosis (ICVAS) classi�cation of congenital vertebral malformation. Reproduced with permission Expert opinion in [7]. Reproduced from Expert Opinion in Expert Opin. Med. Diagn. (2008) 2(10):1107-1121 with permission of Informa UK Ltd. dysostosis, Klippel-Feil, etc.) [54–56], and mode of inher- usage of the term “Jarcho Levin syndrome” has been associ- itance [57]. Recently a proposed pilot classi�cation system ated with a wide range of inconsistent skeletal features, and by the International Consortium for Vertebral Anomalies has been used indiscriminately, ICVAS has recommended (CVM) and Scoliosis (ICVAS) was outlined algorithmically that this term not be used. A high degree of inter observer in Figure 4 [58]. A category of vertebral segmentation defects reliability has been noted with the proposed classi�cation (VSDs) may be de�ned as a single (SVSD) or multiple system, which provides a basis for future cohort genetic (MVSD). Known syndromes such as hemifacial microsomia analysis of similar CVM phenotypes. or VACTERL may be associated with an SVSD. MVSDs are de�ned as generali�ed when there is involvement of �� or greater contiguous vertebral bodies and may represent 6. Monogenic CVM a de�ned phenotype such as spondylocostal dysostosis or spondylothoracic dysostosis, or an unde�ned phenotype. Mutations in �otch signaling genes have been identi�ed in Alternatively, MSVD may have a regional distribution and be two monogenic forms of CVM. Spondylocostal dysostosis associated with a de�ned or unde�ned phenotype. Since prior (SCD) is an autosomal recessive disorder, with occasional Scienti�ca 5

Molecular diagnosis of vertebral segmentation disorders in humans 1 h 30

Rostral SII Somitogenesis SI Somite formation Definition of prospective antero-posterior somitic compartments Boundary specification

Rostral PSM

Tail bud 0 h 1 h 3 h 30 6 h Extension of 9 h the embryo Caudal Caudal 18 h (a) (b)

F 4: Illustration of somite formation from the presomitic mesoderm (PSM) in the chick embryo. Paired somites are formed every 90 minutes in a periodic fashion every 90 minutes shown in (a). (b) A molecular clocked linked to segmentation by dynamic and periodic expression of the cyclic genes in the PSM. Top: Lunatic Fringe mRNA expression appears as a wave sweeping across the whole PSM once during each somite formation as illustrated by in situ hybridization in this 17-somite-old chick embryo. During each somite formations, PSM cells illustrated by the dot undergo a phase of upregulation of the cycling genes followed by a phase of down regulation of these genes. Bottom: As shown in this schematic representation of the progression of somitogenesis in the embryo, the cycles of expression of the cyclic genes will last while the cells remain in the PSM, which corresponds approximately to the time to form 12 somites in the chick embryo. ese PSM cells undergo 12 oscillations of the expression of the cycling genes. Reproduced with permission Expert Opinion in [9]. Reproduced from Expert Opinion in Expert Opin. Med. Diagn. (2008) 2(10):1107-1121 with permission of Informa UK Ltd.

autosomal dominant inheritance. Radiographically, SCD is in Figure 6 [64]. ere is some degree of respiratory com- characterized by contiguous vertebral segmentation defects promise due to the presence of the short thoracic cage. in addition to rib abnormalities Figure 5. Affected individ- STD is caused by mutations in the MESP2 gene, and has a uals have disproportionate short stature, characterized by prevalence of 1/12,000 in the Puerto Rican population, with a shortened trunk and protuberant abdomen. Associated a suggestion of a founder effect of the E103X (p.Glu103X) features include scoliosis and mild respiratory compromise. mutation among Puerto Ricans [65]. Only 25 of affected Mutations in DLL3, a Notch pathway signaling gene, were children with STD survive into adolescence and adulthood, identi�ed in Arab-Israeli and Pakistani kindreds using syn- indicating that the degree of respiratory compromise% is more teny conversion analysis [59]. Mutations in Notch signaling severe in STD as compared to SCD. oracic insufficiency pathway genes, including MESP2 [60], LFNG [61], and HES7, syndrome is associated with STD and is associated with have subsequently been identi�ed [62]. e term “pebble underlying diminished lung volume and chest wall stiffness. beach” sign refers to morphologically abnormal vertebral CVM can be associated with a variety of syndromes as shown bodies characterized by a smooth, round contour, usually in Table 1. associated with the presence of a DLL3 mutation [63]. Hypoplasia of the le vertebral artery has been reported in one affected individual with a compound heterozygous 7. Sporadically Occurring CVMs mutation in HES7 (158D/V186Y). Spondylothoracic dysostosis (STD) is an autosomal Because CVM and associated syndromes usually represent recessive disorder of vertebral segmentation with a clinical sporadic occurrences, even within a particular family, it is phenotype of disproportionate short stature, with increased difficult to identify causal genetic factors. A panel of genes thoracic anterior posterior diameter. STD has a radiographic associated with vertebral patterning defects including PAX1, appearance characterized by the presence of posterior rib DLL3, SLC35A3, WNT3A, TBX6, and T (Brachyury) were fusion, also referred to as a “crab like thorax,” as illustrated sequenced by our group in 50 patients with heterogeneous 6 Scienti�ca

T 1: Some syndromes that include congenital vertebral malformations.

Syndrome OMIM reference Corresponding gene(s) Acrofacial dysostosis 263750 Aicardi ∗ 304050 Alagille∗ 118450 JAGGED1, NOTCH2 Anhalt 601344 Atelosteogenesis∗ III 108721 FLNB Campomelic dysplasia 114290 SOX9 Casamassima-Morton-Nance 271520 Caudal regression ∗ 182940 Cerebro-facio-thoracic∗ dysplasia 213980 CHARGE ∗ 214800 CHD7 “Chromosomal” Currarino 176450 HLXB9 DeLa Chapelle 256050 DeGeorge/Sedlackova∗ 188400 Microdeletion, 10p13-p14, 22q11.2, Dysspondylochondromatosis Femoral hypoplasia-unusual facies∗ 134780 Fibrodysplasia ossi�cans progressive∗ 135100 ACVR1 Fryns-Moerman Goldenhar (Oculo-auriculo-vertebral∗ spectrum) 164210 Incontinentia∗ Pigmenti 308300 NEMO Kabuki 147920 MLL2 Kaufman-McKusick 236700 MKKS KBG Syndrome 148050 Klippel-Feil ∗ 118100 ?PAXl, GDF6 Larsen ∗ 150250 FLNB Lower mesodermal agenesis Maternal diabetes ∗ MURCS Association∗ 601076 Multiple Pterygium Syndrome∗ 265000 CHRNG OEIS Syndrome 258040 Phaver ∗ 261575 Rapadilino∗ 266280 RECQL4 Robinow 268310 ROR2 Rolland-Desbuquois 224400 Rokitansky Sequence∗ 277000 ?WNT4 Silverman ∗ 224410 HSPG2 Simpson-Golabi-Behmel 312870 GPC3 Sirenomelia 182940 Spondylocarpotarsal∗ Synostosis 272460 FLNB Spondylocostal Dysostosis 277300 DLL3, MESP2, LFNG Spondylothoracic Dysotosis 277300 MESP2 akker-Donnai ∗ 227255 Toriello ∗ Urioste ∗ VATER/VACTERL∗ 192350 Verloove-Vanhorick∗ 215850 Wildevanck ∗ 314600 Zimmer ∗ 273395 Underlying∗ cause not known. Reproduced from Expert Opinion in Expert Opin. Med. Diagn. (2008) 2(10):1107-1121 with permission of Informa UK Ltd. ∗ Scienti�ca 7

TBX6, speci�cally rs2289292 (located at exon 8) and rs380962 (locatedatthe5UTR), may have an important role in the pathogenesis of′ congenital scoliosis in the Chinese Han population [72]. CVM may mediated through complex interactions of genetic, environmental and epigenetic factors. Gestational hypoxia in Hes7 and Mesp2 mice results in an increase in severity of CVM+/− in mice. is+/− effect mediated by abnormal FGF signaling results in altered somitogenesis and provides evidence that an environmental trigger such as hypoxia can potentiate a CVM occurrence in a genetically suscep- tible background [73]. e observation that the phenotypic expression of tail kinks in the axin fused mouse can be altered by increased DNA methylation supports𝐹𝐹𝐹𝐹 an epigenetic contribution to CVM occurrence [74]. (𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴 ) Whole exome sequence (WES) and whole genome sequence (WGS) platforms represent suitable platforms for the identi�cation of candidate gene sequence variants F 5: Radiographic features of spondylocostal dystostosis and copy number variants (CNV). WES analyzes approx- including contiguous vertebral malformations with asymmetric rib imately 1 of the entire genome and highlights identi- malformations. Photograph courtesy of Peter D. Turnpenny M.D., Royal Devon and Exeter Hospital. �cation of sequence variation in the coding and splice site regions% in annotated genes identifying approximately 20,000 sequence variants. WGS is capable of uncovering all genetic and genomic variations, including single nucleotide variants (SNV) and CNV identifying approximately 3.5 million sequence variants [75]. A variety of �ltering algo- rithms, including elimination of sequence variants, present in databases such as dbSNP and the 1,000 Genomes Project database, are implemented to narrow down potential can- didate genes. Among coding variants decreasing priority is given to nonsense, frameshi, splice-site, and missense muta- tions. Inheritance modeling (dominant, recessive) computer prediction in conjunction with disease speci�c information helps to enable further re�nement. Evidence for localization of vertebral patterning genes identi�ed in mice, �enopus, and chickens, in synteny blocks supports a hypothesis for conservation of vertebral patterning genes among amniotes [76]. SNV identi�ed in patterning F 6: Radiograph features of spondylothoracic dysostosis genes previously identi�ed in model organisms should be demonstrating contiguous vertebral malformations with symmetric sought initially, although the advantage of WES and WGS is posterior rib fusion. Reproduced with permission Expert Opinion in [9]. the ability to identify novel genes and pathways associated with disease. Following identi�cation of a narrowed and focused list of candidate genes, functional con�rmation is necessary. WES is applicable for the identi�cation of SNV types of CVMs [66–70]. A mutation (c.1013C T) resulting in highly penetrant mendelian disease phenotypes, whereas in an alanine to valine change was found at amino acid WGS has applications for both mendelian and complex position 338 in the T (Brachyury) gene in three affected > phenotype identi�cation in addition to sporadic phenotypes patients, in this cohort that was not present among 886 whicharetheresultofde novo CNVs or SNVs. chromosomes in the CEPH diversity panel [66]. Collectively these patients had maternal pregnancy exposure histories of 8. “Sporadically” Occurring diabetes, valproic acid, and clomiphene. e third affected individual did not have any history of maternal exposure CVM-Related Syndromes during pregnancy. e phenotypes of these patients were all Oculo-auriculo-vertebral spectrum disorders and Klippel- distinct and included cervical and thoracic CVM and sacral Feil syndrome are two frequently encountered syndromes agenesis. is mutation had previously been described in associated with CVM. Progress has been made in under- another individual with sacral agenesis with no history of standing their etiologies and each is discussed below. maternal diabetes during pregnancy [71]. Although no muta- tions in TBX6 were identi�ed in the previously described 8.1. Oculo-Auriculo-Vertebral Spectrum (Hemifacial Micro- patient series, polymorphisms of the somite patterning gene somia). Major clinical features of oculo-auriculo-vertebral 8 Scienti�ca spectrum (OAVS) include unilateral microtia, craniofacial HOXA1, DCC, and GDF6 [89]. Analysis of additional patients asymmetry, mandibular hypoplasia, ocular epibulbar der- would be helpful to support a hypothesis for mutations in moid, and CVM [77]. Additional features include: cle lip genes associated with corticospinal axon path development. with or without cle palate, congenital heart disease, and A mutation at a highly conserved region in the BMP congenital renal malformations. ere is overlap between ligand GDF6 gene c.866T C was identi�ed in both familial OAVS and other syndromes including Treacher Collins and sporadic forms of Klippel Feil syndrome [90]. e vari- syndrome (associated with microtia, lower eyelid colobomas, able expressivity in affected> family members and incomplete and mandibular hypoplasia), Fanconi Anemia (radial ray penetrance observed in GDF6 knockout mice suggest thresh- abnormalities, short stature, elevated diepoxy butane induced olds of GDF6 necessary for spine development are subject to chromosome breakage), and VACTERL syndrome. At the modi�cation by environmental factors and may vary between present time there is no common etiology for OAVS, although individuals and within different spinal regions. An autosomal there is evidence supporting vascular disruption [78], mater- dominant mutation (R266C) in GDF3 has been identi�ed nal diabetes [79], and other teratogenetic agents including in one family with ocular defects including iris and retinal retinoic acid [80] and thalidomide [81]. Using high density coloboma and CVM [91]. �ebra�sh morpholinos for Gdf1/3 oligonucletotide microarray CGH technology, 12 of 86 (14 ) demonstrated retinal colobomas and trunk shortening with patients with hemifacial microsomia studied were identi�ed vertebral malformations. as having a CNV, including 4 patients with deletions and/or% 8 patients with duplications ranging between 2.3–2.8 Mb in size [82]. Of the three patients with CVM who had CNV,one 9. Idiopathic Scoliosis patient had a paternally inherited 9q34.11 duplication. None 9.1. Management. While CS is associated with underlying of the genes involved in the 9q34.11 have any known function CVM, the spine in IS has a normal morphologic appearance. with respect to vertebral body development; a second patient e incidence of IS for treatable curves de�ned as 25 or had a duplication involving 20p12.2. e ANKRD5 gene greater is greater in females than in males with a ratio of 2 : 1, was present within this region and is not known to have ∘ respectively. Gender differences may underlie scoliotic curve any known function in somite formation; the third patient progression [92]. had a coincident isodicentric Y chromosome. ese results Current management of IS in a growing child includes: indicate that CNV represents a minority of genetic causes for (1) Observation of curves that are 25 , (2) Bracing fulltime hemifacial microsomia and support a hypothesis for genetic for curves progressing 25 , and (3) surgery (spinal fusion heterogeneity of OAVS. ∘ and instrumentation) for curves∘ <40–45 . By age 16, 0.6 of affected people will have> required active∘ treatment with a full-time thoracolumbar-sacral orthosis (TLSO) or surgical 8.2. Klippel Feil Syndrome. e majority of cases of Klippel- > % correction with instrumentation [3]. Bracing involves the Feil syndrome (short neck, low posterior hairline, and fusion wearing of a TLSO 22 hours/day until spinal maturation of cervical vertebrae) represent sporadic occurrences within [93]. Fulltime bracing is 80 –85 effective in holding curves a family. However, Klippel-Feil syndrome may represent a under the surgery range at the completion of growth. How- familial occurrence in which multiple family members are ever, in spite of full compliance with brace wear, there is a affected. Autosomal dominant, autosomal recessive, and X- % % 15 –20 failure of bracing, and surgery is indicated. linked forms of Klippel-Feil syndrome have been reported ough scoliosis manifests during adolescence, it contin- [83]. Wildervank syndrome refers to a constellation of fea- ues to cause signi�cant medical problems most of late ado- tures including Klippel-Feil syndrome, congenital hearing % % lescent and adult life. e population of scoliotic teenagers loss, Duane retraction syndrome (limitation of abduction treated in the 1950s and 1960s has now reached adult- with narrowing of the palpebral �ssure and retraction of the hood. ose who underwent surgical corrections are now globe) [84]. manifesting the late effects of both the underlying scoliosis Klippel-Feil syndrome is sometimes associated with mir- and interventional outcomes. ose who had no surgical ror movements, or the involuntary movement of the one intervention also manifest the later effects of scoliosis: back extremity mimicking the opposite extremity, with a central pain, progression, and signi�cant respiratory and cardiac mirror serving as a reference point, re�ecting the image compromise [94]. ese late consequences are not surprising of the voluntary extremity to the opposite side [85–88]. in light of the pathological consequences associated with One neuroanatomic basis for mirror movements is hypoth- the disorder. Signi�cant health problems have been reported esized to be related to variations in the normal pathways of in association with IS, including chronic back and neck descending corticospinal tracts, including the crossed lateral pain, �atback syndrome, disc herniations, osteoarthritis, corticospinal tract (LCT), uncrossed anterior corticospinal osteoporosis, kyphosis, disability, cosmetic dissatisfaction, tract (ACT), and anterolateral corticospinal tract (ALCT) and psychologic distress [95]. Patients with severe scoliosis, [88]. Other hypotheses include delayed resolution following that is, curves 70 , are 3 times more likely to die from a CNS insult or loss of normal control pathways. No coding cardiopulmonary disease than unaffected individuals [96]. mutations were identi�ed in a series of genes associated ∘ with aberrant ocular motor and corticospinal axon path > development in a patient with Wildervanck syndrome, mir- 9.2. Genetic Etiologies of IS. e mode of inheritance of IS ror movements and neuroschisis, including ROBO3, CHN1, has not been solidly established and is under debate [1, 26, Scienti�ca 9

95, 97–100]. Inheritance patterns reported include autosomal this information in combination with additional clinical dominant with variable penetrance, autosomal recessive, information to determine which patients using a series of 52 multifactorial, and X-linked dominant modes. Hypotheses single nucleotide polymorphisms associated with genetic loci advanced to explain pathogenesis of IS include abnormalities on all chromosomes except 3, 13, 21, and the Y chromosome, in the composition of the connective tissue matrix, mela- in conjunction with a the Cobb angle at the time of initial tonin, calmodulin, neuromuscular imbalance, and altered diagnosis, a logistic regression analysis has been utilized to vestibular function. Previous studies, illustrated in Table 2, obtain an AIS Prognostic Test score between 1 and 200 [124]. demonstrated genetic heterogeneity for IS, although no single In three tested populations, low risk scores of 41 were gene linked with the development of IS has been identi�ed to observed to have a negative predictive value of 100 , 99 , date. and 97 . High risk scores (181–200) would identify< the 1- Candidate gene analysis of IS has focused on strati�cation 2 of patients most likely to progress to a severe curve.% ose% of genes on the basis of their presumed function includ- patients% with intermediate risk scores (51–180) would require ing: connective tissue, bone formation and metabolism, close% follow up for their curve progression by an orthope- melatonin signaling pathway, puberty, and growth [115]. dic surgeon. Presently, information regarding the biological Several genes encoding extracellular matrix proteins, includ- function of the genes used for the AIS Prognostic Test score ing elastin, types I and II collagen (COL1A1, COL1A2, is incomplete. e potential advantage of prognostic testing COL2A1), and �brillin, failed to demonstrate linkage to IS would be to reduce costs of imaging in those patients who are [97, 116]. Melatonin is considered a contributor to IS based at a lower risk for scoliosis curve progression. on the observation that pinealectomy in newborn chickens In addition to previous studies suggesting a genetic com- leads to a spinal deformity similar to IS in humans [117]. ponent linked to the development of IS as a binary trait, there Melatonin signaling was also impaired in patients with IS is evidence that genetic factors may predict curve progression [118]. However, no evidence for linkage of IS to chromosome in IS. An association study performed in 304 females with 4q, the locus for the human melatonin 1A receptor, has been IS demonstrated a signi�cantly greater Cobb angle at the observed, indicating that scoliosis does not result solely from time of growth maturation among patients with estrogen melatonin de�ciency [105, 119]. receptor genotype XX and Xx compared to patients with Linkage to 19p13 was described in two separate studies genotype xx [122]. A higher risk for operative [102, 108]. Two loci within this region are credible candidates treatment was observed among patients with genotype XX for IS: �brillin 3 and thromboxane A2 receptor. Fibrillin 3 is and Xx, compared(𝑃𝑃 to patients푃 푃푃푃푃푃푃 with genotype xx. ere is also a component of the extracellular matrix, which contributes evidence that elevated calmodulin levels contribute to curve to micro�brillar structure. Since abnormalities in platelet progression in IS, possibly through interference with estrogen function have been reported in IS [120, 121], attention binding to the estrogen receptor [123]. has turned towards understanding the interaction between e single-nucleotide polymorphism SNP-418G/C in the calmodulin, myosin, and actin in platelets and subsequent tissue inhibitor of metalloproteinase-2 gene promoter region development of IS. was associated with thoracic scoliosis curve severity [125]. ese studies described above were largely based on Downregulation of TIMP-2 transcriptional activity resulting analysis of strategically spaced genetic markers across the in increased vascular proliferation and enhanced anterior genome in large families with IS in order to identify linkages spine endochondral ossi�cation during adolescence could to a chromosomal region corresponding to the potential result in disproportionate spinal growth and result in thoracic genetic basis for IS. Further exploration of candidate gene scoliosis. e promoter polymorphism (rs11063714) in the region(s) demonstrating association with familial IS would be neurotrophin 3 (NTF3) gene is associated with curve severity required to determine their relative contribution to isolated for IS in the Chinese Han population. Individuals affected sporadic (non familial) cases of IS. with IS having an AA genotype had lower mean maximum Cobb angle as compared to patients with AG and GG geno- 9.3. Genetic Prognostic Factors Associated with IS and Curve types [126]. Patients who were skeletally mature and had an Progression. Why and which curves will fail treatment are AA genotype had greater success for treatment with bracing not known. eories abound as to hypokyphotic curves, as compared to patients with GG genotype. Egr 3 mice larger magnitude curves, and less �exible curves. ere fail to express NTF3 and have proprioceptive dysfunction is evidence that genetic factors such as estrogen receptor −/− due to muscle spindle agenesis, apoptosis of proprioceptive genotype may predict curve progression in IS [122]. ere is also evidence that elevated calmodulin levels contribute to neurons, proprioceptive neuron apoptosis, and disruption curve progression in IS, possibly through interference with of synaptic connectivity between muscle sensory and motor estrogen binding to the estrogen receptor [123]. SNPs have neurons. A reduction in the number of muscle spindles been used to develop an AIS-Prognostic Test (AIS-PT) to and malfunction has been demonstrated in spinal muscle identify the curves that will not require bracing or surgery obtained from patients with IS, examined histologically and and curves that will fail bracing. histochemically [127]. ere is also increased expression of Determining which children with adolescent idiopathic NTF3 messenger RNA in paravertebral muscle in IS [119]. scoliosis (AIS) between the ages of 9 and 13 years will ese observations in addition to a strong linkage signal on require bracing is a challenge for the treating orthopedic chromosome 12p13 [111], the NTF3 locus provide support surgeon. An application of genetic knowledge is to use for a role of NTF3 in the pathogenesis of IS. 10 Scienti�ca

T 2: Summary of prior genetic linkage studies for IS.

No. of Study Region(s) Model Comments Families/Individuals Genome wide search in one family of French 6q Acadian and English descent (7 affected Wiseetal.[101] 1/14 distal 10q Autosomal dominant members), with validation of “hot spots” in 18q a second large family Recruited Asian patients in whom scoliosis Chan et al. [102] 7/52 19p13.3 Autosomal dominant developed in adolescence Baghernajad Salehi et 1/17 17p.11 Autosomal dominant 3 generation Italian family al. [103] Maximum lod score of 1.69 (theta = 0.2) Xq23 identi�ed at marker GATA172D05. A lod Justice et al. [104] 202/1198 X-linked dominant Xq26.1 score of 2.23 for this marker was found in one family with six affected individuals Morcuende et al. No linkage to MTNR1A (Melatonin 47/176 4q35 N/A [105] Receptor 1A) and no mutations in MTNR1A Pericentric inversion in chromosome 8 Bashiardes et al. [106] 7 individuals 8p23.2-8q11.21 Autosomal dominant disrupts SNTG1 (syntrophin). Five of 7 individuals in family have SNTG1 deletion Miller et al. [107] 202/1198 6, 9, 16 and 17 Autosomal dominant Model independent linkage analysis reshold of curvature set at 30 . Fibrillin 3, Alden et al. [108] 202/1198 19p11.3 Autosomal dominant thromboxane A2 receptor, possible∘ candidates Baghernajad Salehi et Chromosome 3 Patients’ familial relationships established 1500 individuals Autosomal dominant al. [103] Chromosome 7 through database CHD7 Gene polymorphisms are associated Gao et al. [109] 52 8q N/A with susceptibility to idiopathic scoliosis 9q31.2-q34.2; Ocaka et al. [110] 25/208 Autosomal dominant Con�rmation of 9q [107] 17q25.3-qter All families contribute to recessive model. Autosomal dominant; Raggio et al. [111] 7/48 12p13.3 5/7 families contribute to the dominant autosomal recessive model LOD score 3.86 Gurnett et al. [112] 1/22 18q Autosomal dominant Scoliosis and pectus excavatum 3p26.3 GWAS study. CHL1, DSCAM, CNTNAP2 Sharma et al. [113] 419 N/A ( ) genes involved in axon guidance −8 GWAS study. LBX1 is determinant of dorsal LBX1 Takahashi et al. [114] 1050 𝑃𝑃 푃 푃 푃푃 N/A spinal neurons; altered somatosensory ( ) function −19 𝑃𝑃 푃 푃푃푃푃 푃푃푃 e above summary illustrates the difficulty of iden- proband had congenital scoliosis [128]. In 52 families with tifying causative genes for IS lies in extreme phenotypic IS a signi�cant linkage peak was observed on chromosome and genetic heterogeneity. Future research will need to be 8q12 (multipoint LOD 2.77; ). Over transmission of aimed at improved strati�cation of clinical cases based on the CHD7 associated polymorphism, rs4738824 in patients factors such as age of onset, curve progression, severity, with IS was observed in a cohort𝑃𝑃 of 52푃 푃푃푃푃 families. Substitution responsiveness to bracing, and correlation with mutations in of the A allele of this polymorphism with the G allele is genes identi�ed using next generation sequence platforms predicted to disrupt a possible binding site for caudal-type suchaswholeexomeandwholegenomeanalysis[115]. (cdx) homeodomain-containing transcription factors. Muta- tions in CHD7, a chromeodomain helicase DNA binding protein are associated with CHARGE syndrome (coloboma 10. Relationship between Congenital and of the eye, heart defects, atresia of the choanae, retarda- Idiopathic Scoliosis tion of growth and/or development, genital and/or urinary abnormalities, and ear abnormalities and deafness) [129]. Multiple studies support a common genetic etiology for A hypothesis for the development of idiopathic scoliosis is congenital and idiopathic scoliosis. A family history of IS CHD7 may act postnatally to alter spinal growth during the was observed in 17.3 of 237 families in which an affected adolescent growth spurt. Chd7 in �ebra�sh is expressed in

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